High Attrition Rates in Drug Development and the High Cost of Drugs
In pharmaceutical research, looking for a new drug takes place in three stages: exploration, discovery, and development. In the first stage, the understanding of the disease state is accumulated, a therapeutic target is selected, and a biological screening assay is developed. The discovery stage begins with ‘hits’ finding, where a company's library of compounds is screened for the IC50 value, the concentration of the compound required to displace 50% of a reference ligand from a target receptor. In the course of a year at a large pharmaceutical company, it is not uncommon to have 100,000 to 1,000,000 library compounds tested against a particular target, which is usually a receptor site on a protein molecule. Of the molecules tested for biological activity, about 3000 to 10,000 are found to be active (hits). The initial part of the discovery step is called ‘lead’ generation, where the most promising subset of the hits is selected for further testing. Of the 3000–10,000 potent molecules, about 400 make it to this step. The selection of leads takes into account biopharmaceutic properties of the hits, such as measured aqueous solubility, octanol-water partition coefficients, plasma stability, human serum protein binding, cytochrome P450 inhibition (oxidative metabolism), liver microsome assay (general metabolism), and membrane permeability, using an in vitro cultured-cell model, such as Caco-2. These various tests filter out many molecules with unfavorable biopharmaceutic ADME properties (absorption, distribution, metabolism, and excretion). Most companies perform fast ADME screens in the hits-to-leads transition to aid in “go—no go” decisions. The selected 400 lead compounds are expected to have good in vivo pharmacokinetic (PK) behavior in animal models developed later. But many of the molecules will underperform in laboratory animals, and will be rejected. In lead optimization, the compounds are rigorously tested for in vitro ADME properties, CNS penetration, selectivity against other similar targets, as well as for cytotoxicity. In the final stages of optimization, where rodent in vivo PK measurements are done, metabolic profiles are developed, and additional animal model toxicity tests are performed, about twelve promising ‘candidate’ molecules survive to enter pre-clinical development, where dosage form design and human PK, safety, and effectiveness testing begin. During the subsequent clinical phases, the number of clinical development molecules dwindles down to about one, a considerable and expensive downsizing from the original 400 promising leads.
ADME is the single largest cause of attrition in drug development, accounting for 39% of the failures. Methods which can lower this high attrition rate would benefit the industry by reducing failure rates, the pharmaceutical companies by reducing costs, and consumers by helping to get better drugs to market, in less time.
The in vitro cultured-cell permeability model (e.g., Caco-2) used in the hits-to-leads transition mentioned above is very expensive and technically challenging to automate in high-throughput applications. As a result, many companies use Caco-2 screen mainly in lead optimization, as a mechanistic secondary screen. Other types of permeability measurements, based on artificial membranes, have been considered, with the aim of improving efficiency and lowering costs. PAMPA has risen to that challenge, as a cost-effective primary permeability screen, most often applied to the 3000–10,000 hits. Some companies are considering using the assay to screen whole libraries of 100,000–1,000,000 molecules, in a target-independent effort to ferret out molecules with poor biopharmaceutic properties.
Properties of the Gastrointestinal Tract (GIT) and the Blood-Brain Barrier (BBB)
The in vitro measurement of permeability by the cultured-cell and by the current PAMPA models underestimates the true membrane permeability, due to the effect of the unstirred water layer (UWL) adjacent to the two sides of the membrane barrier. This UWL is 1500 to 2500 μm thick. Transport of lipophilic molecules becomes diffusion-limited in the in vitro assays, and lipophilic molecules all show nearly the same effective permeability. In contrast, the UWL in the human small intestine is about 30–100 μm, and it is virtually zero in the BBB. [Avdeef, A. Curr. Topics Med. Chem. 2001, 1, 277–351] Transport of lipophilic molecules in the GIT is membrane-limited, and values of permeability can be several orders of magnitude higher than predicted from the in vitro assays. Thus, correcting the in vitro permeability data for the UWL effect is important for both GIT and BBB absorption modeling.
The in vivo environment of the GIT is characterized by a pH gradient, with pH 7.4 in the receiving compartment (blood), and pH varying in the donor compartment (lumen) from about 5 to 8 from the start to the end of the small intestine. In contrast, the BBB has pH 7.4 on both sides of the barrier. Modeling the GIT and the BBB requires proper pH adjustment in the in vitro models.
The acceptor compartment in the GIT has a prevailing strong sink condition, made possible by the high concentration of proteins, such as human serum albumin (HSA), circulating in the blood. This affects lipophilic molecules, which can strongly bind to the serum proteins. In contrast, comparable binding of lipophilic molecules takes place on both sides of the BBB and there is an absence of a strong circulation system in the brain fluids. Consequently, in the GIT, lipophilic molecules are swept away from the acceptor site of absorption; in the brain, lipophilic molecules have a greater tendency to accumulate in the BBB, compared to the GIT. In practical terms, the in vitro GIT model calls for a sink condition predominantly in the acceptor compartment; the in vitro BBB absorption model would be served well with comparable sink conditions in both acceptor and donor sides.
In the GIT, about 13% of the phospholipids are negatively charged, with the rest being zwitterionic. The negative charge content is about twice as large in the BBB. Factoring this into the in vitro model is expected to be important.
The ‘white fat’ content of the GIT is higher than that of the BBB. Consequently, the use of triglycerides, cholesterol esters, and cholesterol in the in vitro modeling is thought to be important.
PAMPA (Parallel Artificial Membrane Permeability Assay)
In the early 1960s it was discovered that when a small amount of a phospholipid (2% wt/vol alkane solution) is placed over a pin hole in a thin sheet of plastic suspended in water, a single bilayer (black) lipid membrane (BLM) forms over the hole. Suitable lipids for the spontaneous formation of a BLM are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and others. BLMs have been viewed as useful biological models, although extremely fragile and tedious to make. Efforts to overcome the limitations of the fragile membranes have evolved with the use of membrane supports, made of porous microfilters.
Kansy et al. [Kansy, M., Senner, F., Gubernator, K., J. Med. Chem. 1998, 41, 1007–1010] reported a study of the permeation of drugs across phospholipid-coated microfilters, using a high-throughput assay they called PAMPA (parallel artificial membrane permeability assay). In this method, a ‘sandwich’ is formed from a 96-well microtitre plate and a 96-well hydrophobic filter microtitre plate, such that each composite well is divided into two chambers, separated by the microfilter (hydrophobic Immobilon-P IPVH, 125 μm thick, 0.45 μm pores, 70% porosity) coated with a 10% wt/vol dodecane solution of a commercially-available egg lecithin extract. These investigators were able to relate their measured fluxes to human absorption values with a hyperbolic curve, much like that indicated in Caco-2 screening. The outliers in their assay were molecules known to be actively transported and, therefore, not expected to be modeled by PAMPA.
The PAMPA method has attracted a lot of favorable attention, and has spurred the development of a commercial instrument. [Avdeef, A., Strafford, M., Block, E., Balogh, M. P., Chambliss, W., Khan, I., Eur. J. Pharm. Sci. 2001, 14, 271–280] The system reported by Avdeef and coworkers is an improvement of the Kansy approach, with several novel features, including a way to assess membrane retention, and to correct for unstirred water layer effects, [Avdeef, A., Tsinman, K., U.S. Provisional Patent Application No. 60/178,616, Jan. 28, 2000] along with improvements in sensitivity of the UV method originally used by Kansy. A microfilter-immobilized 2% wt/vol dioleoylphosphatidylcholine (DOPC, a high-purity synthetic phosphatidylcholine) dodecane solution was used as a membrane barrier. The iso-pH permeability equation was introduced, [Avdeef, A., Curr. Topics Med. Chem. 2001, 1, 277–351] which directly takes into account the membrane retention of a drug:
                              P          e                =                              -                                          2.303                ⁢                                                                  ⁢                                  V                  D                                                            A                ⁡                                  (                                      t                    -                                          τ                      SS                                                        )                                                              ⁢                      (                          1                              1                +                                                      V                    D                                    /                                      V                    A                                                                        )                    ⁢                                    log              10                        ⁡                          [                              1                -                                                      (                                                                  1                        +                                                                              V                            A                                                    /                                                      V                            D                                                                                                                      1                        -                        R                                                              )                                    ⁢                                                                                    C                        A                                            ⁡                                              (                        t                        )                                                                                                            C                        D                                            ⁡                                              (                        0                        )                                                                                                        ]                                                          (        1        )            where A=area of filter (cm2), t=time (s), τSS=steady-state time(s), VA and VD are the acceptor and donor volumes (cm3), respectively, and CA(t) and CD(t) are the measured acceptor and donor sample concentrations (mol cm−3) at time t, respectively.The membrane retention factor, R, is defined as1−[CD(t)+CA(t)·VA/VD]/CD(0).The R factor is often stated as a mole percentage (% R) of the sample (rather than a fraction). Its value can at times be very high, as high as 90% for chlorpromazine and 70% for phenazopyridine, when 2% wt/vol DOPC in dodecane is used. Membrane retention is due to the lipophilicity of molecules. Cultured-cell assays also are subject to sample retention by the cell monolayer. Sawada et al. Sawada, G. A., Barsuhn, C. L., Lutzke, B. S., Houghton, M. E., Padbury, G. E., Ho, N. F. H., Raub, T. J., J. Pharmacol. Exp. Ther. 1999, 288, 1317–1326 cited values as high as 89%. This is undoubtedly a common phenomenon with research compounds, which are often very lipophilic.
Batzl-Hartmann et al. [Batzl-Hartmann, C., Hurst, L., Maas, R., German Patent Application: DE 10118725, Oct. 24, 2002; Priority Application DE 2001-10118725, Apr. 12, 2001] claimed an improved PAMPA, where in addition to the normal Kansy procedure, an extra permeablility measurement was made with hydrophilic PVDF filters, where the lecithin did not entirely plug up the microchannels, allowing for aqueous pore diffusion of hydrophilic (but apparently not lipophilic) molecules. The microchannels in the filters appear to get plugged up after the filter microtitre plate is vigorous aggitated. Similar work, although not called ‘PAMPA,’ had been reported by Ghosh, [Ghosh, R., J. Mem. Sci. 2001, 192, 145–154] employing octanol-impregnated cellulose microporous filters, where controlled aqueous pores were formed by applying pressure.
Wohnsland and Faller [Wohnsland, F., Faller, B., J. Med. Chem. 2001, 44, 923–930] modified the PAMPA assay using phospholipid-free hexadecane, supported on 10 μm thick polycarbonate filters (3 μm pores, 20% porosity), and were able to demonstrate interesting predictions. Their PAMPA method (based on UV spectrophtometry in 100–200 μM sample solutions) appears to be an excellent substitute for determining alkane-water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. However, since the alkane membrane barrier is inert, hydrogen-bonding and ionic equilibria, found in natural membrane barriers, cannot be modeled. Apparently, membrane retention was not measured.
Sugano and coworkers [Sugano, K., Hamada, H., Machida, M., Ushio, H., Saitoh, K., Terada, K., Int. J. Pharm. 2001, 228, 181–188] explored the lipid model containing several different phospholipids, resembling the mixture found in reconstituted brush-border lipids, and demonstrated improved property predictions. The best-performing composition consisted of a mixture of five lipids (0.8% PC, 0.8% PE, 0.2% PS, 0.2% PI, 1.0% cholesterol) dissolved in 1,7-octadiene. Apparently, membrane retention was not measured. Concentrations of sample solutions (initial donor concentration of 500 μM) were determined by UV spectrophtometry. Although very promising as a mechanistic probe, the multi-phospholipid mixture is expensive. Also, the use of the volatile octadiene requires an extraction hood for safety reasons.
Zhu et al. [Zhu, C., Jiang, L., Chen, T.-M., Hwang, K.-K., Eur. J. Med. Chem. 2002, 37, 399–407] found the use of hydrophilic filters (low protein binding PVDF) as an advantage in lowering the permeation time to 2 h. Egg lecithin, 1% wt/vol in dodecane, was used as the membrane medium. Concentrations of sample solutions (donor at 100–200 μM at time zero) were determined by UV spectrophtometry. Over 90 compounds were characterized at pH 5.5 and 7.4. For each molecule, the greater Pe value of the two measured at different pH was used to compare to Caco-2 permeabilities reported in the literature. It is noteworthy that many ionizable molecules did not follow the permeability-pH dependency expected from the pH partition hypothesis. It may be that water microchannels (cf., Batzl-Hartmann et al.) were contributing to the unexpected permeability-pH trends. Solute retention by the membrane was not considered. Human intestinal absorption (HIA) values were compared to PAMPA measurements, Caco-2 permeability, octanol-water partition coefficients, calculated polar surface area, and published quantitative structure-property relations. It was concluded that PAMPA and Caco-2 measurements best predicted HIA values.
Permeability Effects of PEG400, Bile Acids, and Other Surfactants
Yamashita et al. [Yamashita, S., Furubayashi, T., Kataoka, M., Sakane, T., Sezaki, H., Tokuda, H., Eur. J. Pharm. Sci. 2000, 10, 109–204] added up to 10 mM taurocholic acid, cholic acid, or sodium laurel sulfate (SLS) to the donor solutions in Caco-2 assays. The two bile acids did not interfere in the transport of dexamethasone. However, SLS caused the Caco-2 cell junctions to become more leaky. The permeability of dexamethasone decreased in SLS. Also, they tested the effect of PEG400, with up to 10% added to donor solutions in Caco-2 assays. PEG400 caused a dramatic decrease (75%) in the permeability of dexamethasone at 10% concentration.
Sugano et al. [Sugano, K., Hamada, H., Machida, M., Ushio, H., Saitoh, K., Terada, K., Int. J. Pharm. 2001, 228, 181–188] also studied the effect of PEG400, up to 30% in both the donor and acceptor wells, in their PAMPA assays. The rationale of using additives was to overcome problems in working with very sparingly soluble compounds. PEG400 dramatically reduced permeability for several of the molecules studied.
In Caco-2 assays, serum proteins had been added to the acceptor compartment, to simulate a sink condition. [Sawada, G. A., Ho, N. F. H., Williams, L. R., Barsuhn, C. L., Raub, T. J., Pharm. Res. 1994, 11, 665–673] Also, serum proteins had been added to the donor solutions in several reports.